1. Field of the Invention
The present invention relates generally to the fields of immunology, virology, and oncology. More particularly, it concerns diagnostic and therapeutic methods related to the development and recurrence of pre-cancerous and cancerous growths or lesions, including cervical intraepithelial neoplasia (CIN), caused by human papilloma virus (HPV).
2. Description of Related Art
Cervical cancer is the second most common malignancy in women worldwide, accounting for 15% of all cancers diagnosed in women (Parkin et al., 1993). In the United States, cervical cancer is one of the most common neoplasm of the female genital tract. Laboratory and epidemiological research has focused on the etiological role of some types of human papilloma virus (HPV) in the pathogenesis of cervical neoplasia (Brinton, 1992; Munoz et al., 1992). Overall, HPV DNA has been detected in more than 79% of specimens of women with definite cervical disease. The most prevalent HPV type is HPV 16, which is detected in high-grade squamous intraepithelial lesions and cancer (Lörincz et al., 1992). Results from epidemiological studies support an association between cervical neoplasia and HPV, which is markedly stronger with HPV type 16 (Morrison et al., 1991; Koutsky et al., 1992; and Munoz et al., 1992). Neoplasia is characterized by abnormal growth of cells, which often results in the invasion of normal tissues, e.g., primary tumors or the spread to distant organs, e.g., metastasis.
The E6 and E7 genes of HPV 16 are frequently co-expressed and are most abundant viral transcripts in biopsies from HPV 16 positive cervical carcinoma (Wettstein, 1990; Seedorf et al., 1987). There is a strong evidence that co-expression of both E6 and E7 open reading frames is necessary and sufficient for efficient malignant transformation of a variety of mammalian cells (Munger et al., 1989). Furthermore, continued expression of the E6 and E7 regions of the viral genome appears to be required to maintain the malignant phenotype (von Knebel Doeberitz et al., 1988).
While some HPV infected patients develop cervical neoplasia, others do not. Also there is a high rate of spontaneous regression observed indicating the role of host immune responses. The induction of a cytotoxic T-lymphocyte (CTL) response constitutes a significant defense mechanism against viral infections; occasionally, a virus-specific CTL response can render full protection without a concomitant antibody response (Sastry et al., 1992; Bevan, 1989; Lukacher, 1984). Based on reports in the literature describing a relation between increased prevalence of anti-HPV antibodies, in particular those directed against the E7 oncoprotein, with severity of the cervical disease (Cason et al., 1992; Hamsikova et al., 1994; Jha et al., 1993), it has been suggested that HPV-specific humoral response may not play a protective role against HPV-associated cervical neoplasia (Nakagawa et al., 1996). On the other hand, it has been reported that individuals with defects in CMI have an increased prevalence of HPV-associated cervical neoplasia, indicating that T cells participate in the control of HPV-associated neoplasia in humans (Nakagawa et al., 1996; Tsukui et al.; 1996; Feltkamp et al., 1993 and Clerici et al., 1997). Decreased IL-2 production and proliferative responses to mitogens such as PHA and concanavalin-A have been observed in patients with invasive cervical carcinoma (Park et al., 1992). A number of in vitro and in vivo strategies have been described to identify peptides from HPV-16 E6, E7, and L1 proteins that induce T-cell activity in mice and humans (Feltkamp et al., 1993; Strang et al., 1990; Tindel et al., 1991; Shepherd et al., 1992; Stauss et al., 1992; Kast et al., 1993). Typically, induction of virus-specific CTLs can be effected by infection with a virus or recombinant virus that expresses a viral gene product. The viral gene product is processed and presented as a peptide on the surface of infected cells in association with an MHC class I molecule for recognition by the CTL (Unanue, 1989).
Additionally, research efforts have concentrated on identifying and characterizing HIV peptides that elicit a viral-specific CTL response. Townsend et al. illustrated the concept of using T-cell epitopes in proteins as vaccine candidates when their group demonstrated the use of short synthetic peptides from influenza nucleoprotein as epitopes for CTL responses (Townsend et al., 1986). The inventors and others have reported using synthetic peptides to generate virus-specific CTLs in vivo (Kast et al., 1991; Aichele et al., 1990; Deres et al., 1989; Sastry et al., 1992; Sastry et al., 1994; Casement et al., 1995) against influenza, lymphocytic choriomeningitis, Sendai virus and HIV.
Over 90% of cervical carcinomas express human papillomavirus (HPV) E6 and E7 proteins. These unique antigens are ideal targets for the development of cytotoxic T-lymphocytes (CTL) for antitumor immunotherapy. Synthetic peptides have been identified corresponding with the E6 and E7 oncoproteins of HPV-16 that were effective in including HPV-specific CTL responses in vivo (Sarkar et al., 1995). Recently, Nakagawa et al. reported that systemic T-cell proliferative responses and CTL responses to HPV-16 peptides and proteins were detectable in many virgin as well as sexually active women without cervical lesions but not in those with active disease (Nakagawa et al., 1997). Similarly, Tsukui et al. reported that TH lymphocyte response, particularly IL-2 production, to HPV antigens was greater among cytologically normal women than in women with different degrees of progressive cervical neoplasia (Tsukui et al., 1996). Also, Clerici et al. observed that production of TH1 cytokines (IL-2 and IFN-γ) which potentially enhances CMI, to be defective in women with extensive HPV infection and that progression to CIN to be associated with a shift from TH1 to TH2 cytokine production (Clerici et al., 1997). Employing a long term in vitro stimulation protocol for determining the TH activity Kadish et al. reported that lymphoproliferative responses to specific HPV peptides were associated with HPV clearance and regression to CIN (Kadish et al., 1997). On the other hand, de Gruijil et al. reported that T-cell proliferative responses to HPV16 E7 peptides correlated with persistence of HPV infection, but antigen-specific IL-2 production was associated with both virus clearance as well as progression of cervical lesions (de Gruijil et al., 1996).
A common clinical management strategy for CIN patients includes excisional or ablative treatment. However, follow-up studies indicate that a significant number of patients experience recurrence. At present no clear understanding exists regarding the development of pre-cancerous or cancerous growths, their recurrence, or disease-free status in the patients who have undergone ablative or excisional treatment for CIN. Better and improved strategies for effective diagnostics of HPV-associated pre-cancerous or cancerous growths and lesions is needed.